Method for activating human antigen presenting cells, activated human antigen - presenting cells, and use thereof

ABSTRACT

The invention relates to a method for activating human-derived antigen-presenting cells by in vitro cultivation with at least one of the glycoside compounds represented by formula (A) or salts thereof [preferred example: (2S,3S,4R)-1-(α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecanediol], and to human antigen-presenting cells activated by the method. The invention also relates to a method for treatment of cancer and infectious diseases including AIDS with the activated human antigen-presenting cells, and to a use of the activated human antigen-presenting cells in the preparation of medicines for treating such diseases. The invention can provide a satisfactory therapeutic effect on cancer and infectious diseases including AIDS without the need to pulse the human antigen-presenting cells with tumor antigens.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel activated humanantigen-presenting cell, a method for activating a humanantigen-presenting cell in vitro, a method for treating cancer andinfectious diseases including AIDS using the activated humanantigen-presenting cell, and a use of the activated humanantigen-presenting cell in preparing medicines for such treatment.

2. Disclosure of Related Art

There have been various strategies for the therapeutical treatment ofcancer patients, including surgical extraction of tumor foci,chemotherapy, radiotherapy and immunotherapy. However, the cure rates ofthese strategies are not as high as they are expected. Therefore, thereexists a strong demand for developing new therapeutic agents and methodswhich can provide improved cure rates in the treatment of cancer.Macrophages, B cells and dendritic cells, which are members ofantigen-presenting cell (APC), have been known to be cells essential forimmune response. Recently, an idea that using APCs, particularlydendritic cells, to induce cancer immunity may be effective in treatingcancer (Grabbe, S. et al., 1995, Immunol. Today, 16, 117) has attractedmuch attention.

Several processes for preparing dendritic cells from mouse have beenknown, such as from the spleen (Clowley, M. et al., 1989, Cell.Immunol., 118, 108), from the bone marrow (Inaba, K. et al., 1992, J.Exp. Med., 176, 1693) and from the epidermis (the epidermis-deriveddendritic cells are known as “Langerhans's cells) (Witmer-Pack, M. etal., 1987, J. Exp. Med., 166, 1487). Furthermore, it was demonstratedthat pulsing the dendritic cells prepared from the murine bone marrowwith tumor antigen and administrating the pulsed dendritic cells into asubject prior to and after the implantation of tumor cells into thesubject can elicit tumor immunity (Celluzzi, C. M. et al., 1996, J. Exp.Med., 183, 283; Paglia, P. et al., 1996, J. Exp. Med., 183, 317).

The following processes are known for preparing human APCs, particularlydendritic cells, from peripheral blood or umbilical cord blood: aprocess in which FcR⁺ cells, T cells, B cells and NK cells are removedfrom human peripheral blood using the antibodies against them fromperipheral blood monocytes to give APCs (Hsu et al., 1996, Nature Med.,2, 52); and a process in which adherent cells in human peripheral bloodmonocytes from which CD19⁺ B cells and CD2⁺ T cells were removed arecultured with a granulocyte-macrophage colony stimulating factor(GM-CSF) and interleukin 4 (IL-4) for about one week (Sallusto, F. etal., 1994, J. Exp. Med., 179, 1109). It has been reported that suchhuman peripheral blood-derived APCs have both allogeneic and autologousMLR (mixed leukocyte reaction) enhancing effects. On the other hand, forpreparing APCs from human umbilical cord blood or bone marrow cells, aprocess is known where GM-CSF and tumor necrosis factor-α (TNF-α) areused to prepare APCs from CD34⁺ cells found in umbilical cord blood orbone marrow (Santiago-Schwartz, F. et al., 1992, J. Leukocyte Biol., 52,274; Caux, C. et al., 1992, Nature, 360, 258). However, although theAPCs prepared from human umbilical cord blood or bone marrow cells bysuch a process as mentioned above have an allogeneic MLR enhancingeffect, they have no (Santiago-Schwartz, F. et. al.) or, if any,extremely poor (Caux et al.) antologous MLR enhancing effect.

Recently, it has been demonstrated that a therapy with the APCs that hadbeen pulsed with tumor antigens is effective for treating B celllymphoma patients (Hsu, F. J. et al., 1996, Nature Med., 2, 52). Thatis, a therapy for B cell lymphoma patients was successfully achieved byculturing APCs prepared from peripheral blood of a B cell lymphomapatient together with B cell lymphoma antigens in vitro andadministrating the cultured APCs into the B cell lymphoma patient.

As shown in this exemplary therapy, methods in which APCs are pulsedwith tumor antigens may be very effective for treating cancer if thecancer can be clearly identified by their tumor antigens, like B celllymphoma. However, such methods as mentioned above take enormous timeand costs, because the tumor antigens are generally specific to theindividual patients and, therefore, must be specified for the respectivepatients, and the specified tumor antigens must be produced in largequantities to pulse the APCs. Furthermore, since it is impossible formost of the cancer patients to identify their tumor antigens, the rangeof application of the method in which APCs are pulsed with tumorantigens is limited. In view of the above, in order to apply the therapywith APCs (hereinafter referred to as “the APC therapy”) recognized tobe a very effective therapy for cancer, to as many patients as possible,there has been a strong demand for development of a method for preparinghuman APCs effective for treating cancer without the aid of tumorantigens.

Within a living body, various types of β-galactosylceramides andβ-glucosylceramides are present, each of which has a sugar linked to aceramide via a β-linkage (Svennerholm, L. et al., 1972, Biochem.Biophys. Acta., 280, 626; Karlsson, K. A. et al., 1973, Biochim.Biophys. Acta., 316, 317). On the other hand, the inventors have foundthat α-galactosylceramides have remarkable immunostimulatory propertiesand anti-tumor properties (Morita, M. et al., 1995, J. Med. Chem., 38,2176), and that such properties of α-galactosylceramides andα-glucosylceramides are far more potent than those of the correspondingβ-anomers (Motoki, K. et al., 1995, Biol. Pharm. Bull., 18, 1487). Itwas also found that compounds having an α-glycosylceramide structureshow a protecting effect against radiation when administered into aliving subject (Motoki, K. et al., 1995, Bioorg. Med. Chem. Lett., 5,2413) and cause an increase in the number of platelets and white bloodcells (Motoki, K. et al., 1996, Biol. Pharm. Bull., 19, 952). Theinventors also have found that an α-galactosylceramide, KRN7000, canenhance the functions of dendritic cell-rich APCs prepared from themurine spleen, and that the enhanced APCs exhibit an anti-tumor effectwhen administered into a subject before tumors are implanted into thesubject (Koezuka, Y. et al., 1995, The 9^(th) International Congress ofImmunology, Abstract, 55; Yamaguchi, Y. et al., 1996, Oncol. Res. 8,399).

However, the effect of glycoside compounds, such as KRN7000, or saltsthereof on APCs rich in dendritic cells from murine bone marrows andLangerhans's cells from murine epidermis has not been clarified. Inaddition, there is no report on anti-tumor effects of the APCs rich inmurine spleen- and bone marrow-derived dendritic cells, which have beenstimulated with KRN7000, upon administration into a subject after tumorsare implanted into the subject. No effect of glycoside compounds such asKRN7000 or salts thereof on human APCs has also been known.

To address the above-mentioned demands, the present invention provides anovel human APC activating agent which is useful in preparing activatedhuman APCs that exhibit a sufficient therapeutic effect on cancer andvarious infectious diseases including AIDS without the need to pulse theantigen-presenting cells with tumor antigens, and also provides a methodfor activating human APCs with such an activating agent.

SUMMARY OF THE INVENTION

The present invention provides a method for activating human APCs, whichcomprises culturing human-derived APCs in vitro with at least one ofglycoside compounds represented by formula (A) or salts thereof:

wherein R₁ to R₉ and X are to be defined later.

According to one embodiment, the present invention provides a method foractivating human APCs which comprises culturing human-derived APCs invitro with a glycoside compound(2S,3S,4R)-1-(α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecanediolor a salt thereof.

The present invention also provides activated human APCs which can beprepared by culturing human-derived APCs in vitro with at least one ofthe glycoside compounds represented by formula (A) above or saltsthereof.

The present invention further provides a method for treating cancer andinfectious diseases including AIDS, which comprises applying an APCtherapy with such activated human APCs as defined above.

The present invention further provides a use of such activated humanAPCs as defined above in the preparation of a medicine for treatingcancer and infectious diseases including AIDS by application of the APCtherapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amount of ³H-TdR taken up into the cells variouslytreated as described. In FIG. 1, “V-APC” and “KRN-APC” refer to humanperipheral blood-derived APCs pretreated with a vehicle and KRN7000,respectively.

FIG. 2 shows a relationship between the number of APCs and the amount of³H-TdR taken up into an allogeneic T cells. In FIG. 2, “V-APC” and“KRN-APC” refer to human umbilical cord blood-derived APCs (CD1c⁺ cells)stimulated with a vehicle and KRN7000, respectively.

FIG. 3 shows a relationship between the number of APCs and the amount of³H-TdR taken up into autologous T cells. In FIG. 3, “V-APC” and“KRN-APC” refer to the same APCs as those used in FIG. 2.

FIG. 4 shows a relationship between the number of APCs and the amount of³H-TdR taken up into spleen cells. In FIG. 2, “V-APC”, “KRN-APC”,“583-APC”, “517-APC”, “564-APC”, “563-APC” and “562-APC” refer to murinespleen-derived APCs pretreated with a vehicle, KRN7000, AGL-583,AGL-517, AGL-564, AGL-563 and AGL-562, respectively.

FIG. 5 shows the survival periods of EL-4-bearing mice when murinespleen-derived APCs pretreated with a vehicle or KRN7000 were injectedintravenously to the mice.

FIG. 6 shows a relationship between the number of APCs and the amount of³H-TdR taken up into T cells. In FIG. 6, “V-APC” and “KRN-APC” refer tomurine bone marrow-derived APCs pretreated with a vehicle and KRN7000,respectively.

FIG. 7 shows the weight of the liver of a tumor-bearing mouse when theV-APC or KRN-APC prepared in the same manner as in FIG. 6 was injectedintravenously into the mouse.

FIG. 8 shows a relationship between the number of APCs and the amount of3H-TdR taken up into spleen cells. In FIG. 8, “V-APC” and “KRN-APC”refer to murine epidermis-derived APCs pretreated with a vehicle andKRN7000, respectively.

FIG. 9 shows the volume of a subcutaneous tumor for each of thetumor-bearing mice when the APCs pretreated with a vehicle, avehicle+B16-tumor cell lysate, KRN7000 or KRN7000+B16-tumor cell lysate(V-APC, V-T-APC, KRN-APC or KRN-T-APC, respectively), respectively, wasinjected intravenously to the mice.

FIG. 10 shows a relationship between the number of APCs given afterculturing for 3 days and the amount of ³H-TdR taken up into humanperipheral blood-derived FcR⁻ cells. In FIG. 10, “V-APC”, “KRN-APC”,“V-APC-G-M” and “KRN-APC-GM” refer to human peripheral blood-derivedAPCs pretreated with a vehicle, KRN7000, a vehicle+GM-CSF+IL4 andKRN+GM-CSF+IL4, respectively.

FIG. 11 shows a relationship between the number of APCs given afterculturing for 7 days and the amount of ³H-TdR taken up into humanperipheral blood-derived FcR⁻ cells. In FIG. 11, “V-APC-GM” and“KRN-APC-GM” refer to the same APCs as those used in FIG. 10.

FIG. 12 shows a relationship between the number of APCs given afterculturing for 11 days and the amount of ³H-TdR taken up into humanperipheral blood-derived FcR cells. In FIG. 12, “V-APC-GM” and“KRN-APC-GM” refer to the same APCs as those used in FIG. 10, and “MCM⁺”and “MCM⁻” respectively mean that the APCs are cultured with and withouta monocyte conditioned medium (MCM).

FIG. 13 shows a relationship between the number of APCs given afterculturing for 3 days and the amount of ³H-TdR taken up into humanumbilical cord blood-derived FcR⁻ cells. In FIG. 13, “V-APC-GM” and“KRN-APC-GM” refer to the human umbilical cord blood-derived APCsrespectively pretreated with a vehicle+GM-CSF+IL4 andKRN7000+GM-CSF+IL4.

FIG. 14 shows activation effect of murine spleen-derived APCs when theyare stimulated by Compounds Nos. 1, 2, 5, 10 and 16 (except KRN7000),termed AGL506, AGL514, AGL571, AGL512 and AGL525, respectively.

DETAILED DESCRIPTION OF THE INVENTION

As a result of extensive and intensive studies by the inventors on theabove-mentioned problems, it was found that the glycoside compoundsaccording to the present invention (representatively, KRN7000) cansignificantly enhance the functions of human APCs prepared from humanperipheral blood and human umbilical cord blood. The glycoside compoundscan also significantly enhance the functions of dendritic cell-rich APCsfrom a murine bone marrow and Langerhans's cell-rich APCs from murineauricle, as well as dendritic cell-rich APCs from a murine spleen. Itwas also found that a significant anti-tumor effect can be observed whendendritic cell-rich APCs from a murine spleen or a murine bone marrowwhich have been cultured in vitro with the glycoside compounds of thepresent invention (representatively, KRN7000) are injected into a mouseinto which tumor cells have been implanted. Based on these findings, theinvention was completed.

That is, the present invention relates to an activating agent (i.e., anantigen-presenting function enhancing agent) comprising a specificglycoside compound, which is useful for preparing activated human APCshaving a therapeutic effect on cancer and infectious diseases includingAIDS. Specifically, the present invention relates to an activating agentfor APCs prepared from human peripheral blood, human umbilical cordblood, human bone marrow cells or human epidermis (i.e., APCs to beactivated). More specifically, the present invention relates to anactivating agent for human monocytes, human dendritic cells or humanCD1c⁺ cells.

According to the present invention, there is provided a method foractivating human antigen-presenting cells which comprises culturinghuman-derived antigen-presenting cells in vitro with at least one of theglycoside compounds represented by formula (A) or salts thereof:

wherein:

R₁ is H or OH;

X is an integer of from 7 to 25;

R₂ is a substituent defined by any one of the following (a) to (e):

-   -   (a) —CH₂(CH₂)_(Y)CH₃;    -   (b) —CH(OH)(CH₂)_(Y)CH₃;    -   (c) —CH(OH)(CH₂)_(Y)CH(CH₃)₂;    -   (d) —CH═CH(CH₂)_(Y)CH₃; and    -   (e) —CH(OH)(CH₂)_(Y)CH(CH₃)CH₂CH₃;    -   wherein Y is an integer of from 5 to 17;

R₃ is H;

one of R₅ and R₆ is H and the other is OH,

one of R₇ and R₈ is H and the other is OH,

In a preferred embodiment, the present invention provide a method foractivating human antigen-presenting cells which comprises using at leastone of the glycoside compounds represented by formula (B) or saltsthereof:

wherein:

R₁, X and R₂ are as defined as in the case of formula (A); and

R₃ to R₉ are substituents defined by any one of the following (i) to(iii):

-   -   (i) [galactose type]    -   each of R₃, R₆ and R₈ is H;    -   (ii) [glucose type] OH        -   each of R₃, R₆ and R₇ is H;        -   R₄, R₅ and R₉ are as defined as in (i) and    -   (iii) [allose type]        -   each of R₃, R₅ and R₇ is H;        -   each of R₄, R₆ and R₈ is OH; and        -   R₉ is H, CH₃ or CH₂OH.

The glycoside compounds defined by formula (A) or (B) above arecomprised of a sugar moiety and an aglycone moiety, and some of them arealso referred to as α-cerebrosides, α-glycosylceramides,α-glucosylceramides, α-galactocerebrosides or a galactosylceramides.These compounds are characterized by having the α-form of anomericconfiguration.

In the glycoside compound, the sugar moiety is preferably of [galactosetype] as defined in (i), and more preferably of one wherein each of R₃,R₆ and R₈ is H, each of R₄, R₅ and R₇ is OH and R₉ is CH₂OH (i.e.,α-galactopyranosyl).

In the glycoside compound, the aglycone moiety preferably has R₂ beingany one of the substituents (b) (c) and (e) above, and more preferablyhas R₁ being H (i.e., kerasin type) and R₂ being the substituent (b). Xis preferably an integer of 21 to 25 and Y is preferably an integer of11 to 15.

Preferable examples of the glycoside compound of the present inventionare listed below. In the list, compounds (1)-(9), (10)-(24), (25)-(31),(32)-(33), and (34) are those compounds in which R₂ is the substituent(a), (b), (c), (d) or (e) above, respectively. The alphabet letters A,B, C and D behind the compounds, name indicate the referencespecifications of WO93/05055, WO94/02168, WO94/09020 and WO94/24142,respectively, which describe the synthesis methods of the annotedcompounds. Among the glycoside compounds below, compound (14)(2S,3S,4R)-1-((α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecanediol(referred to as “KRN7000 hereinbelow), is most preferable. With respectto this compound, an example of the synthesis process will beillustrated in the Production Example and Scheme 1 below. (1)(2S,3R)-1-(α-D-galactopyranosyloxy)-2-[(R)-2- Ahydroxytetracosanoylamino]-3-octadecanol (2)(2S,3R)-1-(α-D-galactopyranosyloxy)-2- Atetracosanoylamino]-3-octadecanol (3)(2S,3R)-1-(α-D-galactopyranosyloxy)-2- Atetradecanoylamino-3-octadecanol (4)(2S,3R)-1-(α-D-glucopyranosyloxy)-2- C tetradecanoylamino-3-octadecanol(5) (2S,3R)-1-(6′-deoxy-α-D-galactopyranosyloxy)-2- Ctetradecanoylamino-3-octadecanol (6)(2S,3R)-1-(β-L-arabinopyranosyloxy)-2- Ctetradecanoylamino-3-octadecanol (7)(2S,3R)-1-(α-D-galactopyranosyloxy)-2- Atetradecanoylamino-3-hexadecanol (8)(2R,3R)-1-(α-D-galactopyranosyloxy)-2- Atetradecanoylamino-3-hexadecanol (9)(2R,3S)-1-(α-D-galactopyranosyloxy)-2- Atetradecanoylamino-3-hexadecanol (10)(2S,3S,4R)-1-(α-D-galactopyranosyloxy)-2-[(R)-2- Ahydroxytetracosanoylamino]-3,4-octadecanediol (11)(2S,3S,4R)-1-(α-D-galactopyranosyloxy)-2-[(R)-2- Ahydroxytetracosanoylamino]-3,4-undecanediol (12)(2S,3S,4R)-1-(α-D-galactopyranosyloxy)-2-[(R)-2- Ahydroxyhexacosanoylamino]-3,4-icosanediol (13)(2S,3S,4R)-1-(α-D-galactopyranosyloxy)-2-[(S)-2- Ahydroxytetracosanoylamino]-3,4-heptadecanediol (14)(2S,3S,4R)-1-(α-D-galactopyranosyloxy)-2- Productionhexacosanoylamino-3,4-octadecanediol Example (15)(2S,3S,4R)-1-(α-D-galactopyranosyloxy)-2- Boctacosanoylamino-3,4-heptadecanediol (16)(2S,3S,4R)-1-(α-D-galactopyranosyloxy)-2- Atetracosanoylamino-3,4-octadecanediol (17)(2S,3S,4R)-1-(α-D-galactopyranosyloxy)-2- Atetracosanoylamino-3,4-undecanediol (18)(2S,3S,4R)-1-(α-D-galactopyranosyloxy)-2- Chexacosanoylamino-3,4-octadecanediol (19)O-β-D-galactofuranosyl-(1→3)-O-α-D- Dgalactopyranosyl-(1→1)-(2S,3S,4R)-2-amino-N-[(R)-2-hydroxytetracosanoyl]-1,3,4-octadecanetriol (20)O-α-D-galactopyranosyl-(1→6)-O-α-D-glucopyranosyl- D(1→1)-(2S,3S,4R)-2-amino-N-hexacosanoyl-1,3,4- octadecanetriol (21)O-α-D-galactopyranosyl-(1→6)-O-α-D- Dgalactopyranosyl-(1→1)-(2S,3S,4R)-2-amino-N-hexacosanoyl-1,3,4-octadecanetriol (22)O-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1 D→1)-(2S,3S,4R)-2-amino-N-hexacosanoyl-1,3,4- octadecanetriol (23)O-(N-acetyl-2-amino-2-deoxy-α-D-galactopyranosyl-(1 D→3)-O-[α-D-glucopyranosyl-(1→2)]-O-α-D-galactopyranosyl-(1→1)-(2S,3S,4R)-2-amino-N-[(R)-2-hydroxyhexacosanoyl-1,3,4-octadecanetriol (24)O-(N-acetyl-2-amino-2-deoxy-α-D-galactopyranosyl-(1 D→3)-O-[α-D-glucopyranosyl-(1→2)]-O-α-D-galactopyranosyl-(1→1)-(2S,3S,4R)-2-amino-N-[(R)-2-hydroxytetracosanoyl-1,3,4-hexadecanediol (25)(2S,3S,4R)-1-(α-D-galactopyranosyloxy)-2-[(R)-2- Ahydroxytricosanoylamino]-16-methyl-3,4-heptadecanediol (26)(2S,3S,4R)-1-(α-D-galactopyranosyloxy)-2-[(S)-2- Ahydroxytetracosanoylamino]-16-methyl-3,4-heptadecanediol (27)(2S,3S,4R)-1-(α-D-galactopyranosyloxy)-16-methyl-2- Atetracosanoylamino]-3,4-heptadecanediol (28)O-β-D-galactofuranosyl-(1→3)-O-α-D- Dgalactopyranosyl-(1→1)-(2S,3S,4R)-2-amino-N-[(R)-2-hydroxytetracosanoyl]-17-methyl-1,3,4-octadecanetriol (29)O-β-D-galactofuranosyl-(1→3)-O-α-D- Dgalactopyranosyl-(1→1)-(2S,3S,4R)-2-amino-N-[(R)-2-hydroxytetracosanoyl]-15-methyl-1,3,4-hexadecanediol (30)O-(N-acetyl-2-amino-2-deoxy-α-D-galactopyranosyl-(1 D→3)-O-[α-D-glucopyranosyl-(1→2)]-O-α-D-galactopyranosyl-(1→1)-(2S,3S,4R)-2-amino-N-[(R)-2-hydroxyhexacosanoyl-16-methyl-1,3,4-octadecanetriol (31)O-(N-acetyl-2-amino-2-deoxy-α-D-galactopyranosyl-(1 D→3)-O-[α-D-glucopyranosyl-(1→2)]-O-α-D-galactopyranosyl-(1→1)-(2S,3S,4R)-2-amino-N-[(R)-2-hydroxytetracosanoyl-16-methyl-1,3,4-heptadecanetriol (32)(2S,3S,4E)-1-(α-D-galactopyranosyloxy)-2- Aoctadecanoylamino-4-octadecene-3-ol (33)(2S,3S,4E)-1-(α-D-galactopyranosyloxy)-2- Atetradecanoylamino-4-octadecene-3-ol (34)(2S,3S,4R)-1-(α-D-galactopyranosyloxy)-2-[(R)-2- Ahydroxypentacosanoylamino]-16-methyl-3,4-octadecanediol

The glycoside compound defined by formula (A) or (B) may form an acidaddition salt with a pharmaceutically acceptable acid. Examples of theacid to be used for formation of such an acid addition salt includeinorganic acids such as hydrochloric acid, sulfuric acid, nitric acidand phosphoric acid; and organic acids such as acetic acid, propionicacid, maleic acid, oleic acid, palmitic acid, citric acid, succinicacid, tartaric acid, fumaric acid, glutamic acid, pantothenic acid,lauryl sulfonic acid, methanesulfonic acid and phthalic acid.

The human APC-activating agent according to the present invention can beused to activate the APCs prepared from various human tissues such asperipheral blood, umbilical cord blood, bone marrow cells and epidermis.Typically, the activating agent of the present invention can be added invitro during or after the preparation of the human APCs (for example,from human peripheral blood, umbilical cord blood or bone marrow cellsand, if necessary, with GM-CSF and IL-4) to give the activated humanAPCs. The activated human APCs may be prepared more efficiently byadding substance(s) such as SCF, IL-1, IL-3, TNF-a and Flk2/Flt3 ligandor a monocyte conditioned medium (MCM) in addition to GM-CSF and IL-4.Moreover, in the preparation of the activated human APCs, tumorantigens, viral antigens or antigen-specific peptides thereof can beadded to give the activated human APCs that are more effective fortreating cancer and infectious diseases.

The human APC-activating agent of the present invention may be dissolvedin an appropriate dissolving solvent such as DMSO and then diluted to anappropriate concentration with physiological saline, a culture medium orthe like. The final concentration of the activating agent in a suitablecultivation for activating human APCs is within the range from 0.1 ng/mLto 10 μg/mL, and preferably from 0.01 μg/mL to 1 μg/mL. It is preferablethat the human APC-activating agent according to the present inventionbe prepared and added immediately before it is used.

Accordingly, the present invention also provides activated human APCswhich can be prepared by culturing human-derived APCs in vitro with atleast one of the glycoside compounds represented by formula (A) or (B)defined above or salts thereof.

In an embodiment of the present invention, the human-derived APCs areCD1c-positive or CD1c-rich cells. Such cells may be prepared from, butnot limited to, human peripheral blood, human umbilical cord blood,human bone marrow cells, human epidermis, and the like.

The use of the human APCs activated with the activating agent of thepresent invention leads to effective APC therapy for patients sufferingfrom cancer. Furthermore, it is expected that the cytotoxic Tlymphocytes (CTLs) prepared by co-culturing the activated APCs andperipheral blood from a patient can be used for an effective therapy forpatients suffering from cancer or infectious diseases including AIDS.

Accordingly, the present invention also provides a method for treatingcancer and infectious diseases including AIDS with the activated humanAPCs.

The present invention further provides a use of the activated human APCsin the preparation of medicines for treating cancer and infectiousdiseases including AIDS by application of an APC therapy.

The following examples illustrate the present invention more clearly;however, these examples are not be construed to limit the scope of theinvention.

EXAMPLES Production Examples

Synthesis of Compound G1

To a solution of D-lyxose (200 g, 1.33 mol) in acetone (3.0 L) driedover calcium chloride was added sulfuric acid (0.5 mL). The mixture wasstirred at room temperature for 18 hours, and Molecular Sieves 4A powder(100 g) was then added thereto. After neutralizing, the reactionsolution was filtered with celite and the residue was washed withacetone. The filtrate and the washings were combined and concentratedunder reduced pressure, thereby giving a crude product of Compound G1 inan amount of 240 g (yield 95%). For the subsequent reaction, the crudeproduct was used without any further purification. For the preparationof a sample for analyses, a portion of the crude product was subjectedto further purification by chromatography on a silica gel column withhexane:acetone (9:1) as an eluent.

mp 76-78° C.; FDMS m/z 191 (M+1)⁺; ¹H-NMR (500 MHz, CDCl₃) δ5.45 (1H, d,J=1.8 Hz), 4.83 (1H, dd, J=3.7, 5.5 Hz), 4.64 (1H, d, J=6.1 Hz),4.27-4.30(1H, m), 3.90-3.99(2H, m) 1.48 (3H, s), 1.32 (3H, s).

Synthesis of Compound G2

To a solution (168 mL) of Compound G1 (239 g, about 1.26 mol) inmethylene chloride were added pyridine (10 mL) and trityl chloride (39.0g), and the mixture was stirred at 32° C. for 4 hours. Ethanol (8 mL)was then added to the solution dropwise and stirred at room temperaturefor 2 hours. The solution was washed with a saturated aqueous ammoniumchloride solution, a saturated aqueous sodium hydrogencarbonate solutionand the brine, and was concentrated under reduced pressure. The residuewas dissolved in ethyl acetate and then cooled to 0° C. to causeprecipitation, thereby giving a product of Compound G2 in an amount of501 g (yield based on the amount of D-lyxose: 87%).

mp 174-176° C.; FDMS m/z 432 M⁺; ¹H-NMR (500 MHz, CDCl₃) δ7.21-7.49(15H, m), 5.38 (1H, d, J=2.4 Hz), 4.75 (1H, dd, J=3.7, 6.1 Hz), 4.59(1H, d, J=6.1 Hz), 4.31-4.35 (1H, m), 3.43 (1H, dd, J=4.9, 9.8 Hz), 3.39(1H, dd, J=6.7, 9.8 Hz), 1.29 (3H, s), 1.28 (3H, s)

Synthesis of Compound G3

To a solution (1500 mL) of tridecanetriphenylphosphonium bromide (962 g,1.16 mol; a product prepared by heating 1-bromotridecane andtriphenylphosphine at 140° C. for 4.5 hours) in THF was added a 2.5 Msolution of n-butyl lithium in hexane (462 mL, 1.16 mol) dropwise in anatmosphere of argon at 0° C. After the dropwise addition was completed,the resultant solution was stirred for 15 minutes. To the solution wasadded a solution (450 mL) of Compound G2 (250 g, 579 mmol) in THFdropwise. The resultant solution was stirred for 18 hours whilegradually elevating the solution temperature to room temperature. Thereaction solution was concentrated under reduced pressure. To theresidue was added a mixed solution of hexane:methanol:water (10:7:3,1000 mL) and washed with a saturated aqueous ammonium chloride solution.The aqueous layer was extracted with hexane (500 mL) several times. Allof the organic layers obtained were combined, dried over anhydrousmagnesium sulfate, and then concentrated under reduced pressure, therebygiving a crude product of Compound G3 in an amount of 339 g (yield:98%). For the subsequent reaction, the crude product was used withoutany further purification. For the preparation of a sample for analyses,a portion of the crude product was subjected to further purification bychromatography on a silica gel column with hexane:ethyl acetate (9:1) asan eluent.

FDMS m/z 598M⁺; ¹H-NMR (500 MHz, CDCl₃) δ 7.21-7.45 (15H, m),5.48-5.59(2H, m), 4.91(0.7H, t, J=7.3 Hz), 4.44(0.3H, t, J=7.3 Hz),4.26(0.3H, dd, J=4.3, 7.3 Hz), 4.21(0.7H, dd, J=4.3, 6.7 Hz), 3.75(0.7H,m), 3.69(0.3H, m), 3.24(0.3H, dd, J=4.9, 9.8 Hz), 3.17(0.7H, dd, J=4.9,9.8 Hz), 3.09-3.14 (1H, (3.11, dd, J=4.9, 9.2 Hz), H1bE overlapped)1.75-2.03(2H, m), 1.49(3H, s), 1.39 and 1.38(3H, each s), 1.21-1.34(20H,m), 0.88(3H, t, J=6.7 Hz).

Synthesis of Compound G4

To a solution (1500 mL) of Compound G3 (338 g, about 565 mmol) inmethylene chloride was added pyridine (500 mL). Subsequentlymethanesulfonyl chloride (49 mL, 633 mmol) was added dropwise theretoand then stirred at 31° C. for 24 hours. Ethanol (40 mL) was addeddropwise thereto and stirred at room temperature for 1 hour. After thereaction solution was concentrated under reduced pressure, a mixedsolution of hexane:methanol:water (10:7:3, 1000 mL) was added to theresidue to cause separation of the solution into an aqueous layer and anorganic layer. The aqueous layer was extracted with hexane (200 mL) 3times. All of the organic layers obtained were combined, dried overanhydrous magnesium sulfate, and then concentrated under reducedpressure, thereby giving a crude product of Compound G4 in an amount of363 g (yield: 95%). For the subsequent reaction, the crude product wasused without any further purification. For the preparation of a samplefor analyses, a portion of the crude product was subjected to furtherpurification by chromatography on a silica gel column with hexane:ethylacetate (9:1) as an eluent.

FDMS m/z 676M⁺; ¹H-NMR (500 MHz, CDCl₃) δ 7.21-7.47 (15H, m), 5.41(0.7H, ddd, J=5.5, 9.2, 11.0 Hz), 5.32(0.7H, bt, J=11.0 Hz), 5.22 (0.3H,bdd, J=9.2, 15.0 Hz), 5.02(0.3H, dt, J_(t)=7.3 Hz, J_(d)=15.0 Hz), 4.8(0.7H, ddd, J=3.1, 5.5, 7.9 Hz), 4.73(0.7H, dd, J=5.5, 9.8 Hz),4.64-4.67 (0.3H, m), 4.61(0.3H, dd, J=5.5, 9.2 Hz), 4.48(0.7, dd, J=5.5,7.9 Hz), 4.22(0.3H, dd, J=5.5, 9.2 Hz) 3.55(0.3H, dd, J=2.4, 11.6 Hz),3.45(0.7H, dd, J=3.2, 11.0 Hz), 3.06-3.12(4H, (3.12,S), (3.11,S), (3.09,dd, J=3.1, 11.0 Hz)), 1.66-1.82 (2H, m), 1.47 and 1.46 (3H, each s),1.39 (3H, s), 1.13-1.35 (20H, m), 0.88 (3H, t, J=6.8 Hz).

Synthesis of Compound G5

To a solution (1500 mL) of Compound G4 (362 g, about 536 mmol) inmethylene chloride was added methanol (350 mL). Concentratedhydrochloric acid (200 mL) was added to the solution dropwise and thenstirred at room temperature for 5 hours. The reaction solution wasneutralized with sodium hydrogencarbonate and then subjected tofiltration. The filtrate was concentrated under reduced pressure, andethyl acetate was added to the residue and then washed with brine. Theaqueous layer was extracted with ethyl acetate several times. All of theorganic layers obtained were combined, dried over anhydrous magnesiumsulfate, and then concentrated under reduced pressure. The resultantproduct was crystallized from hexane to give a product of Compound G5 inan amount of 161 g (yield based on the amount of Compound G2: 70%).

mp 66-67° C.; FDMS m/z 377(M−H₂O)+; ¹H-NMR(500 MHz, CDCl₃+D₂0) δ5.86(0.3H, dt, J_(t)=7.3 Hz, J_(d)=14.7 Hz), 5.77(0.7H, dt, J_(t)=7.3,J_(d)=10.4 Hz), 5.55(0.3H, br.dd, J=7.3, 14.7 Hz), 5.49(0.7H, bt, J=9.8Hz), 4.91-4.97(1H, m), 4.51 (0.7H, bt, J=9.8 Hz), 4.11(0.3H, bt, J=7.3Hz), 3.94-4.03 (2H, m), 3.67-3.73 (1H, (3.70, dd, J=3.1, 6.7 Hz), (3.69,dd, J=3.1, 7.3 Hz)], 3.20 and 3.19 (3H, each s), 2.05-2.22(2H, m),1.22-1.43(20H, m), 0.88(3H, t, J=6.7 Hz).

Synthesis of Compound G6

To a solution (780 mL) of Compound G5 (160 g, 405 mmol) in THF was added5% palladium on barium sulfate (16 g). After the reaction vessel waspurged with hydrogen gas, the mixture was stirred at room temperaturefor 20 hours. The reaction solution was filtered with celite and thefilter cake was washed with a mixed solution of chloroform:methanol(1:1). The filtrate and the washings were combined and concentratedunder reduced pressure. The residue was crystallized from ethyl acetateto give a product of Compound G6 in an amount of 146 g (yield: 91%).

[α]₂₃ ^(D)+12^(O)(c 1, CHCl₃/MeOH=1:1); mP 124-126° C.; FDMS m/z 397(M+1)⁺; ¹H-NMR (500 MHz, CDCl₃/CD₃=1:1) δ 4.93-4.96(1H, m, H2), 3.91(1H,dd, J=6.7, 12.2 Hz), 3.85 (1H, dd, J=4.9, 12.2 Hz), 3.54-3.60(1H, m),3.50(1H, dd, J=1.8, 8.5 Hz), 3.19(3H, s), 1.75-1.83(1H, m),1.53-1.62(1H, m), 1.2-1.45(24H, m), 0.89(3H, t, J=6.7 Hz).

Synthesis of Compound G7

To a solution (1000 mL) of Compound G6 (145 g, 365 mmol) in DMF wasadded sodium azide (47 g, 730 mmol), and the mixture was stirred at 95°C. for 4 hours. The reaction solution was concentrated. Ethyl acetate(450 mL) was added to the residue, and the resultant solution was washedwith water. The aqueous layer was re-extracted with ethyl acetateseveral times. All of the organic layers obtained were combined, washedwith brine, dried over anhydrous magnesium sulfate and then concentratedunder reduced pressure, thereby giving a crude product of Compound G7 inan amount of 122 g (yield: 97%). For the subsequent reaction, the crudeproduct was used without any further purification. For the preparationof a sample for analyses, a portion of the crude product was subjectedto further purification by chromatography on a silica gel column withhexane:ethyl acetate (9:1) as an eluent.

[α]₂₃ ^(D)+16.5^(O)(c 0.5, CHCl₃/MeOH =1:1); mP 92-93° C.; FDMS m/z344(M+1)⁺; ¹H-NMR (500 MHz, CD₃OD) δ 3.91(1H, dd, J=3.7, 11.6 Hz),3.75(1H, dd, J=7.9, 11.6 Hz), 3.49-3.61 (3H, m), 1.50-1.71(2H, m),1.22-1.46(24H,m), 0.90(3H, t, J=6.7 Hz)

Synthesis of Compound G8

To a solution (750 mL) of Compound G7 (121 g, about 352 mmol) inmethylene chloride were added pyridine (250 mL) and trityl chloride (124g, 445 mL), and the mixture was stirred at room temperature for 16hours. Ethanol (30 mL) was added dropwise thereto and then stirred atroom temperature for 30 minutes. After washing with a saturated sodiumhydrogencarbonate solution, a saturated aqueous ammonium chloridesolution and brine, the reaction solution was dried over anhydrousmagnesium sulfate and then concentrated under reduced pressure. Theresidue was purified by chromatography on a silica gel column withhexane:ethyl acetate (10:1) as an eluent, thereby giving a product ofCompound G8 in an amount of 34.4 g (yield based on the amount ofCompound G6: 52%).

[α]₂₄ ^(D)+11.9^(O)(c 0.9, CHCl₃), FDMS m/z 585 M⁺; ¹H-NMR (500 MHz,CDCl₃+D₂0) δ 7.24-7.61(15H, m), 3.62-3.66(2H, m), 3.51-3.57(2H, m),3.42(1H, dd, J=6.0, 10.4 Hz), 1.23-1.56(26H, m), 0.88(3H, t, J=6.7 Hz).

Synthesis of Compound G9

To a solution (300 mL) of Compound G8 (33.5 g, 57.3 mmol) in DMF wasadded 60% hydrogenated sodium (5.5 g, about 138 mmol in terms of NaH),and the mixture was stirred for 40 minutes. The reaction solution wascooled to 0° C., and then benzyl chloride (15 mL, 120 mmol) was addeddropwise thereto. The resultant solution was stirred for 18 hours whilegradually elevating the solution temperature to room temperature.Ice-cooled water (100 mL) was added to the reaction solution to stop thereaction, and the reaction solution was then extracted with ethylacetate. The extract was washed with brine 3 times. All of the organiclayers obtained were combined, dried over anhydrous magnesium sulfate,and then concentrated under reduced pressure, thereby giving a crudeproduct of Compound G9 in an amount of 42.2 g (yield: 96%). For thesubsequent reaction, the crude product was used without any furtherpurification. For the preparation of a sample for analyses, a portion ofthe crude product was subjected to further purification bychromatography on a silica gel column with hexane:ethyl acetate (100:1)as an eluent.

[α]₂₄ ^(D)+9.8^(O)(c 1.0, CHCl₃), FDMS m/z 738 (M−N₂)+; ¹H-NMR (500 MHz,CDCl₃) δ 7.07-7.48(25H, m), 4.57(1H, d, J=11.6 Hz), 4.44(1H, d, J=11.6Hz), 4.41 (2H, s), 3.73-3.79 (1H, m), 3.46-3.56 (2H, m), 3.37 (1H, dd,J=8.6, 10.4 Hz), 1.20-1.64 (26H, m), 0.88 (3H, t, J=6.7 Hz).

Synthesis of Compounds G10 and G11

To a solution (250 mL) of Compound G9 (41.2 g, about 54 mmol) in1-propanol was added methanol (30 mL), and subsequently 5% palladium oncarbon (4.1 g) and ammonium formate (27.1 g, 4.3 mol) were addedthereto. After the mixture was stirred at room temperature for 16 hours,it was diluted with ethyl acetate and then filtered with celite. Thefiltrate was concentrated under reduced pressure and then dissolved inethyl acetate. The resultant solution was washed 3 times with asaturated aqueous sodium hydrogencarbonate solution and subsequentlywith brine. All of the organic layers obtained were combined, dried overanhydrous magnesium sulfate, and then concentrated under reducedpressure, thereby giving a crude product of Compound G10 in an amount of38.9 g (98%). For the subsequent reaction, the crude product (CompoundG10) was used without any further purification.

To a solution (300 mL) of Compound G10 in methylene chloride were addedhexacosanic acid (22.4 g, 56.5 mmol) and WSC hydrochloride (12.6 g, 64.6mmol), and the mixture was refluxed with heating for 2 hours. Aftercooling to room temperature, the reaction solution was concentratedunder reduced pressure. Ethyl acetate (500 mL) was added to the residue,and the resultant solution was washed several times with a 0.5 M aqueoussolution of hydrochloric acid, brine, a saturated aqueous sodiumhydrogencarbonate solution and then brine. All of the organic layersobtained were combined, dried over anhydrous magnesium sulfate, and thenconcentrated under reduced pressure, thereby giving a crude product ofCompound G11 in an amount of 53.2 g (yield: 88%). For the subsequentreaction, the crude product (Compound G11) was used without any furtherpurification. For the preparation of a sample for analyses, a portion ofthe crude product was subjected to further purification bychromatography on a silica gel column with hexane:ethyl acetate (100:1)as an eluent.

[α]₂₄ ^(D)+5.3^(O)(c 0.4, CHCl₃); FDMS m/z 1118 M⁺; ¹H-NMR (500 MHz,CDCl₃) δ 7.20-7.38(25H, m), 5.57(1H, d, J=9.1 Hz), 4.80 (1H, d, J=11.6Hz), 4.48-4.50 (3H, m), 4.24-4.32(1H, m), 3.83 (1H, dd, J=3.0, 6.7 Hz),3.43-3.51(2H, m, H1a), 3.29(1H, dd, J=4.3, 9.8 Hz), 1.92(2H, t, J=7.3Hz), 1.28-1.60(72H, m), 0.88(6H, t, J=6.7 Hz).

Synthesis of Compound G12

To a solution (180 mL) of Compound G11 (52.2 g, about 47 mmol) inmethylene chloride was added methanol (36 mL), and a 10% solution ofhydrochloric acid in methanol (3.0 mL) was added dropwise thereto. Themixture was stirred at room temperature for 2 hours. The reactionsolution was neutralized with sodium hydrogencarbonate powder (18 g) andthen filtered with celite. The residue was washed with methylenechloride. The filtrate and the washings were combined, and then washedwith brine. The organic layer obtained was dried over anhydrousmagnesium sulfate, and then concentrated under reduced pressure. Theresidue was dissolved in hot acetone and then cooled to 0° C. to causeprecipitation of a product of Compound G12 in a purified form in anamount of 38.6 g (yield based on the amount of G9: 77%).

[α]₂₄ ^(D)-29.7^(O)(c 0.7, CHCl₃); mp 75-76.5° C.; FDMS m/z 876M⁺;¹H-NMR (500 MHz, CDCl₃) δ 7.30-7.47(10H, m), 6.03(1H, d, J=7.9 Hz), 4.72(1H, d, J=11.6 Hz), 4.66(1H, d, J=11.6 Hz), 4.61(1H, d, J=11.6 Hz),4.45(1H, d, J=11.6 Hz), 4.12-4.17(1H, m), 4.00(1H, dt, J_(t)=4.3,J_(d)=7.3 Hz), 3.67-3.72(2H, m), 3.61(1H, ddd, J=4.3, 8.6, 11.6 Hz),1.94-2.05(2H, m), 1.15-1.69(72H, m), 0.88(6H, t, J=6.1 Hz).

Synthesis of Compound G13

(1) 2,3,4,6-Tetra-O-benzyl-D-galactopyranosyl acetate (79.8 g) wasdissolved in a mixed solution of toluene (160 mL) and isopropyl ether(520 mL) and then cooled to −10-0° C. To the solution was added asolution containing 2.0 equivalent amount of HBr in isopropyl ether (2.8mmol/mL, about 100 mL). After the reaction solution was stirred at−10-0° C. for about 90 minutes, a 5% aqueous solution of sodiumhydrogencarbonate was poured thereto and stirred to neutralize theexcess HBr. The whole of the solution was transferred to a separatoryfunnel to cause separation into an aqueous layer and an organic layer.The aqueous layer obtained was discarded and the organic layer waswashed with a 10% aqueous solution of sodium chloride twice. The organiclayer was concentrated under reduced pressure, thereby giving a syrupproduct of 2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl bromide (Gal-Br).

(2) To a solution (420 mL) containing Compound G12 (60.0 g, 68.6 mmol),tetrahexylammonium bromide (89.4 g, 206 mmol) and Molecular Sieves 4Apowder (60 g) in toluene were added DMF (140 mL) and subsequently asolution (250 mL) of Gal-Br (about 137 mmol) in toluene. The mixture wasstirred at room temperature for 72 hours. Methanol (12 mL) was added tothe reaction solution and stirred for 2 hours. After the solution wasfiltered with celite, the filtrate was washed with a saturated aqueoussodium hydrogencarbonate solution and brine, dried over anhydrousmagnesium sulfate, and then concentrated under reduced pressure.Acetonitrile was added to the residue, and the resultant solution wasstirred for 2 hours to cause precipitation. The precipitate obtained wasdried under reduced pressure to give a dry powder product. The powderproduct was purified by chromatography on a silica gel column withhexane:ethyl acetate (8:1) as an eluent, thereby giving Compound G13 inan amount of 70.9 g (yield: 74%).

[α]₂₄ ^(D)+18.8^(O)(c 0.9, CHCl₃); mp 74-75° C.; FDMS m/z 1399 M+1)⁺;¹H-NMR (500 MHz, CDCl₃) δ 7.21-7.37(30H, m), 6.12 (1H, d, J=9.0 Hz),4.91(1H, d, J=11.6 Hz), 4.84(1H, d, J=3.7 Hz), 4.72-4.80 (4H, m),4.35-4.65 (7H, m), 4.12-4.18 (1H, m), 3.99-4.05(2H, m), 3.84-3.93 (4H,m), 3.73(1H, dd, J=3.7, 11.0 Hz), 3.47-3.51(2H, m), 3.42(1H, dd, J=6.1,9.1 Hz), 1.87-1.99(2H, m), 1.18-1.70(72H, m), 0.88(6H, t, J=7.4 Hz).

(2S, 3R,4R)-1-O-(α-D-Galactopyranosyl)-N-hexacosanoyl-2-amino-1,3,4-octadecanetriol(KRN7000)

Compound G13 (60.0 g, 42.9 mmol) was suspended into ethanol (960 mL),and a 20% suspension of palladium hydroxide (6.0 g) in ethanol was addedthereto. 4-Methylcyclohexene (120 mL, 93.5 mmol) was added to thesuspension as a donor compound for hydrogen, and then refluxed underheating for 4 hours. Thereafter, the resultant solution was filtered toremove the catalyst. The residue was washed with hot ethanol. Thefiltrate was allowed to stand at room temperature to cause precipitationof a white product. The white precipitate was filtered out and thendried under reduced pressure. The powder product obtained was suspendedinto ethanol:water (92:8, 3.5 L), dissolved by heating while stirring,and then allowed to stand to cause precipitation again. The precipitatesolution was filtered and the filter cake obtained was dried underreduced pressure, thereby giving the title compound in a white powderform in an amount of 35.0 g (yield: 95%).

[α]₂₄ ^(D)+43.6^(O)(c 1.0, pyridine); mp 189.5-190.5° C.; negative FABMSm/z 857(M−H)⁻; IR(cm⁻¹, KBr) 3300, 2930, 2850, 1640, 1540, 1470, 1070;¹H-NMR(500 MHz, C₅D₅N) δ 8.47(1H, d, J=8.5 Hz), 5.58(1H, d, J=3.7 Hz),5.27(1H, m), 4.63-4.70 (2H, m), 4.56(1H, m), 4.52(1H, t, J=6.1 Hz),4.37-4.47 (4H, m), 4.33(2H, m), 2.45(2H, t, J=7.3 Hz), 2.25-2.34 (1H,m), 1.87-1.97(2H, m), 1.78-1.85(2H, m), 1.62-1.72(1H, m),1.26-1.45(66H,m), 0.88(6H, t, J=6.7 Hz). ¹³C-NMR (125 MHz, C₅D₅N) δ173.2(s), 101.5(d), 76.7(d), 73.0(d), 72.5(d), 71.6(d), 71.0(d),70.3(d), 68.7(t), 62.7(t), 51.4(d), 36.8(t), 34.4(t), 32.1(t), 30.4(t),30.2(t), 30.03(t), 30.00(t), 29.93(t), 29.87(t), 29.81(t), 29.76 (t),29.6(t), 26.5(t), 26.4(t), 22.9(t), 14.3(q).

Pharmacological Tests

[Pharmacological Test 1] Effort of KRN 7000 on Human PeripheralBlood-Derived APC

This experiment was designed to study an effect of KRN7000 (CompoundNo.14, a representative glycoside compound of the invention) on APCsprepared from human peripheral blood. The APCs (mainly comprised ofdendritic cells) derived from human peripheral blood were preparedessentially according to the method of Hsu et al. (supra). Briefly,human peripheral blood was layered over Ficoll-Paque and centrifuged at2,000 rpm for 35 minutes to give a mononuclear cell fraction. A portionof the mononuclear cell fraction was suspended into PBS (Ca⁺⁺, Mg⁺⁺free) supplemented with 5% of inactivated human AB serum. The resultantsolution was layered over 50% Percol, and then centrifuged at 3,000 rpmfor 20 minutes to give a high-density fraction. The high-densityfraction was panned using a human γ-globulin-coated dish to remove FcR⁺cells therefrom (i.e., a monocyte-removed fraction). The resultantfraction was reacted with anti-CD3 monoclonal antibodies, anti-CD21monoclonal antibodies and anti-CD56 monoclonal antibodies. Afterwashing, the resultant solution was panned using an anti-mouse IgGantibody-coated dish to remove cells that were reactive with the aboveantibodies (i.e., T cells, B cells and NK cells). The cell fraction thusobtained (i.e., a fraction from which monocytes, T cells, B cells and NKcells were removed) was cultured in RPMI 1640 medium supplemented with5% of inactivated human AB serum in the presence of a vehicle (DMSO,final concentration: 0.1%) and KRN7000 (final concentration: 100 ng/mL)for 40 hours. After washing, the resultant cells were provided for useas APCs.

On the other hand, as responder cells, CD4⁺ T cells were purified fromthe rest of the above-prepared mononuclear cell fraction using a humanCD4⁺ T cell-enrichment column, and were cultured in RPMI 1640 mediumsupplemented with 5% of inactivated human AB serum until they weresubjected to use with the APCs.

The APCs (5×10⁴ cells/well) and the responder cells (1×10⁵ cells/well)both prepared in the above steps were added to wells of a 96-wellflat-bottomed plate, and then subjected to autologous MLR assay. Duringthe last 12 hours of the 6-day culture period, tritium-thymidine(³H-TdR) was added to each of the wells (0.5 μCi/well), and then thecells were harvested. The amount of the 3H-TdR taken up into the cellswas determined using a liquid scintillation counter. The results (meanvalue and standard deviation of 3 wells) are shown in FIG. 1. In FIG. 1,“V-APC” and “KRN-APC” refer to the APCs pretreated with the vehicle andKRN7000, respectively.

As shown in FIG. 1, when each of CD4⁺ T cell, the vehicle-pretreated APC(V-APC) and the KRN7000-pretreated APC (KRN-APC) were cultured singly,in either case, the cells took up no or a negligible amount of ³H-TdR.In contrast, when the above-prepared T cell was mixed with V-APC, anobvious ³H-TdR uptake was observed. When the T cell was mixed withKRN-APC, a significantly remarkable ³H-TdR uptake was observed. Theseresults demonstrate that KRN-APC has a more significant autologous MLRenhancing effect than V-APC. Accordingly, KRN7000 has an activatingeffect on the APCs prepared from human peripheral blood (i.e., anantigen-presenting function enhancing effect).

(Pharmacological Test 2] Effect of KRN7000 on Human Umbilical CordBlood-Derived APC

APCs derived from human umbilical cord blood (CD1c positive cells; CD1c⁺cells) were prepared in the following manner. That is, human umbilicalcord blood was layered over Lymphoprep and centrifuged at 1500 rpm for20 minutes to give a mononuclear cell fraction. A portion of themononuclear cell fraction was washed with PBS supplemented with 2%inactivated FBS and 1 mM EDTA twice, and FcR was blocked with humanγ-globulin. The solution was reacted with anti-CD1c antibodies, andwashed with PBS supplemented with 2% FBS and 1 mM EDTA twice. Thesolution was further reacted with anti-mouse IgG microbeads, washed withPBS supplemented with 2% FBS and 1 mM EDTA, and then suspended into PBSsupplemented with 0.5% BSA and 5 mM EDTA. CD1c⁺ cells were obtained fromthe suspension using a MiniMACS magnetic column previously washed withPBS supplemented with 0.5% BSA and 5 mM EDTA. The CD1c⁺ cells obtainedwere suspended into RPMI 1640 medium supplemented with 10% inactivatedhuman AB blood serum to adjust its density to 1×10⁶ cells/mL. The cellsuspension was added to wells of a 24-well plate (2 mL/well), and then avehicle (DMSO, final concentration: 0.1%) or KRN7000 (finalconcentration: 100 ng/mL) was added to each of the wells. Afterincubating for 3 days, the plate was washed 3 times and provided for useas APCs.

CD4⁺ T cells were purified from the rest of the above-preparedmononuclear cell fraction using a human CD4⁺ T cell-enrichment column.The T cells obtained were suspended into RPMI 1640 medium supplementedwith 10% inactivated human AB blood serum to adjust its density to 1×10⁶cells/well. The suspension was added to wells of a 24-well plate (2mL/well) and cultured for 3 days. The T cells thus obtained were used asresponder cells for autologous MLR assay. On the other hand, aperipheral blood was collected from a normal volunteer, and CD4⁺ T cellswere purified therefrom using a human CD4⁺ T cell-enrichment column. TheT cells thus obtained was used as responder cells for allogeneic MLRassay. The responder cells (1×10⁵ cells/50 mL/well) and the stimulatorcells (0, 1.000, 10000 cells/50 mL/well) were added to wells of a96-well flat-bottomed plate, and the plate was incubated. For allogeneicMLR assay, the plate was incubated for 4 days, ³H-TdR was added to eachof the wells (0.5 μCi/well). After 20 hours, the cells were harvestedand the amount of the ³H-TdR taken up into the cells was determinedusing a liquid scintillation counter. The results (mean value andstandard deviation of 3 wells) are shown in FIG. 2.

For autologous MLR assay, the plate was incubated for 7 days, ³H-TdR wasadded to each of the wells (0.5 μCi/well). After 8 hours, the cells wereharvested and the amount of the ³H-TdR taken up into the cells wasdetermined using a liquid scintillation counter. The results (mean valueand standard deviation of 3 wells) are shown in FIG. 3. In FIGS. 2 and3, “V-APC” and “KRN-APC” refer to the APCs (CD1c⁺ cells) pretreated withthe vehicle and KRN7000, respectively.

As shown in FIG. 2., the CD1c⁺ cells stimulated with the vehicle (APCs;V-APC) promoted the uptake of ³H-TdR (proliferation of allogeneic Tcells) in a manner dependent on the number of the V-APC, whereas theCD1c⁺ cells stimulated with KRN7000 (APCs; KRN-APC) showed a strongerallogeneic T cell-proliferation-promoting effect than V-APC.

As shown in FIG. 3, when autologous T cells were used as the respondercells, the CD1c⁺ cells stimulated with the vehicle (V-APC) hardlystimulated the proliferation of the autologous T cells even if the APCswere added to the wells in an amount of 10000 cells/well; whereas theCD1c cells treated with KRN7000 (KRN-APC) significantly stimulated theproliferation of the autologous T cells in a manner dependent on thenumber of the APCs, similar to the case for the allogeneic T cells.

[Pharmacological Test 3] Effect of α- and β-galactosylceramides and α-and β-glucosylceramides on Murine Spleen-Derived APC

The preparation of dendritic cell-rich APCs from a murine spleen wasperformed essentially in accordance with the method of Clowley, M. etal. (supra). Briefly, the spleen was removed from a BDF1 mouse. Afterthe treatment with 100 U/mL of collagenase, the spleen was dissectedwith tweezers to separate into a cell suspension and a tissue debris.The tissue debris was suspended into a 400 U/mL solution of collagenase.After culturing in a CO₂ incubator for 20 minutes, the suspension waspassed through a stainless steel mesh with an inner syringe to give acell suspension. Two of the cell suspensions were combined, andsubjected to centrifugation. The cell suspension was subjected todensity gradient centrifugation with a high-density BSA to give alow-density cell fraction. The cell fraction was plated onto a 60 mmdish and cultured for 2 hours. The procedure for removing the floatingcells was repeated 3 times. RPMI 1640 medium supplemented with 10%inactivated FBS was added to the dish and subsequently any one ofKRN7000 (Compound No.14), AGL583 (a β-anomer of KRN7000), AGL517(Compound No.3), AGL564 (a β-anomer of AGL517), AGL563 (Compound No.4),AGL562 (a β-anomer of AGL-563) and a vehicle (DMSO, final concentration:0.1%) was also added thereto to a final concentration of 100 ng/mL.After culturing overnight, the non-adherent cells were collected, andthe resultant cells were provided for use as APCs.

On the other hand, BDF1 mouse spleen cells were treated with a red bloodcell-lysing buffer comprised of NH₄Cl and Tris-HCl to hemolyze red bloodcells, and the resultant spleen cells were suspended into RPMI 1640medium supplemented with 10% inactivated FBS. The spleen cells wereinoculated onto a 100 mm dish, and cultured in a CO₂ incubator for 2hours. Thereafter, the non-adherent cells were recovered, which wereprovided for use as responder cells.

The above-prepared APCs (1×10^(3,) 3.3×10³ or 1×10⁴ cells/well) and theresponder cells (2.5×10⁵ cells/well) were added to wells of a 96-wellplate for syngeneic MLR assay. After culturing for 1 day, ³H-TdR wasadded to each of the wells (0.5 μCi/well). After 16 hours, the cellswere harvested, and the amount of ³H-TdR taken up into the cells wasdetermined using a liquid scintillation counter. The results (mean valueand standard deviation of 3 wells) are shown in FIG. 4.

In FIG. 4, “V-APC”, “KRN-APC”, “583-APC”, “517-APC”, “564-APC”,“563-APC” and “562-APC” refer to the APCs pretreated with the vehicle,KRN7000, AGL-583, AGL-517, AGL-564, AGL-563 and AGL-562, respectively.

As shown in FIG. 4, the APCs pretreated with α-galactosylceramides suchas KRN-APC and 517-APV showed a significant syngeneic MLR enhancingeffect, whereas the APCs pretreated with β-galactosylceramides such as583-APC and 564-APC did not show any such effect. The APCs pretreatedwith α-glucosylceramides such as 563-APC also showed a remarkablesyngeneic MLR enhancing effect, whereas the APCs pretreated withβ-glucosylceramides such as 562-APC did not show any such effect.

[Pharmacological Test 4] APC Therapy With Murine Spleen-Derived APCsPretreated With KRN7000

To study an anti-tumor effect of murine spleen-derived APCs activatedwith KRN7000 on mice implanted with tumor cells, experiments wereperformed with BDF1 mice (6-week-old, female). All experiments included6 mice per group. Murine T cell lymphoma EL-4 cells were injectedintravenously into each mouse at a level of 1×10⁵ cells/mouse (injectionday: day 0). On day 1, the vehicle-pretreated APCs (V-APC) and theKRN7000-pretreated (100 ng/mL) APCs (KRN-APC) both prepared in the samemanner as in Pharmacological Test 3 were each injected intravenouslyinto the mice at a level of 5×10⁵ cells/mouse. As a positive control,KRN7000 was injected intravenously into the mice at a dose of 100 μg/kgon days 1, 5 and 9. Each mouse was checked for a sign of life daily todetermine its survival period. The results are shown in FIG. 5.

As shown in FIG. 5, when V-APC was injected, no prolongation of survivalperiod was observed. However, when KRN-APC was injected, a remarkableincrease in survival period was observed and 50% of the KRN-APC-injectedmice were cured completely. When KRN7000 was injected intravenously at adose of 100 μg/kg 3 times, a remarkable increase in survival period wasobserved, however, it was found that the potency of the effect wasweaker than that when KRN-APC was injected only once.

(Pharmacological Test 5] Antigen Presenting Function Enhancing Effort ofKRN7000 on Murine Bone Marrow-Derived Dendritic Cell

Dendritic cell-rich APCs derived from murine bone marrow were preparedaccording to the method of Inaba, K. et al. (supra) with somemodifications (Yamaguchi Y. et al., Stem Cells, 1977:15:144-153).Briefly, bone marrow cells from BALB/c mice were prepared. Afterhemolyzing the red blood cells with a NH₄Cl solution, FcR⁺ cells wereremoved by panning using a human γ-globulin-coated dish. The cells thusobtained were suspended in RPMI medium supplemented with 10% FCS, andcultured in the presence of 10 ng/mL of mouse rGM-CSF and 10 ng/mL ofhuman rTGF-β for 6 days at 5×10⁵ cells/well (1 ml/well, a 24-wellplate). Every 2 days, each well was washed briefly using a Pasteurpipette and then about 75% of the medium was sucked out and supplementedwith 1 ml of a fresh medium containing the above factors. Afterculturing for 6 days, non-adherent cells were recovered, and FcR⁺ cellswere removed therefrom by panning using a human γ-globulin-coated dish.The cells thus prepared were cultured in a culture medium supplementedwith 10 ng/mL of mouse rGM-CSF and 10 ng/mL of human rTNF-α foradditional 2 days. During this culture, either a vehicle (DMSO, finalconcentration: 0.1%) or KRN7000 (final concentration: 100 ng/mL) wasadded to the culture medium. The cells were collected and washed 3 timesto provide for use as APCs. On the other hand, as responder cells, Tcells were prepared from a BALB/c murine spleen using a T cellenrichment column (R&D). T cells were added to wells of a 96-well plate(3×10⁵ cells/well), and the above-prepared APCs were further addedthereto for syngeneic MLR assay (3×10⁴, 1×10⁴, 3×10³ and 1×10³cells/well, respectively). After culturing for 2 days. ³H-TdR was addedto each of the wells (0.5 μCi/mL). After 6 hours, the cells wereharvested and determined on the uptake of ³H-TdR into the cells using aliquid scintillation counter. The results (mean value and standarddeviation of 3 wells) are shown in FIG. 6. In FIG. 6, “V-APC” and“KRN-APC” refer to the APCs pretreated with the vehicle and KRN7000,respectively.

As shown in FIG. 6, the murine bone marrow-derived APCs which werestimulated with KRN7000 (KRN-APC) showed a remarkable syngeneic MLRenhancing effect compared to the APCs stimulated with the vehicle(V-APC).

(Pharmacological Test 6] APC Therapy With Murine Bone MarrowDerived-APCs Pretreated with KRN7000

The experiment was performed to study an anti-tumor effect of murinebone marrow-derived APCs activated with KRN7000 on mice implanted withtumor cells. All experiments were performed with CDF1 mice (6-week-old,female), which were divided into groups consisting of 5 mice. Murinecolon adenocarcinoma Colon26 cells were implanted into each mouseintrasplenically at a level of 2×10⁶ cells/mouse (inoculation day: day0). On day 1, the vehicle-pretreated APCs (V-APC) and theKRN7000-pretreated (100 ng/mL) APCs (KRN-APC) both prepared in the samemanner as in Pharmacological Test 5 were individually injectedintravenously to the mice at a level of 8×10⁵ cells/mouse. As a positivecontrol, KRN7000 was injected intravenously to the mice at a dose of 100μg/kg on days 1, 5 and 9. On day 14, the liver was removed from each ofthe mice and weighed. The results (mean value and standard deviation ofthe weights of the liver of 5 mice) are shown in FIG. 7. Here, the liverfrom the mouse that had not been implanted with the tumor cells wasabout 1 g. Accordingly, the substantial tumor weight was determined bysubtracting 1 g from the weight of the liver of the tumor-implantedmouse.

As shown in FIG. 7, when V-APC was injected, a slight suppressive effecton tumor growth was observed; whereas when KRN-APC was injected, aremarkable inhibitory effect on tumor growth was observed and no tumornodule in the liver was observed by naked eyes in 3 of the 5 mice. WhenKRN7000 was injected intravenously 3 times at a dose of 100 μg/kg, aremarkable inhibitory effect on tumor growth was observed, which was atthe same level as that when KRN-APC was injected once.

[Pharmacological Test 7] Antigen Presenting function Enhancing Effect ofKRM7000 on Murine Epidermis-Derived APCs

Langerhans's cell-rich APCs were prepared from murine auricleessentially in accordance with the method of Witmer-Pack, M. et al.(supra). Briefly, the epidermis of auricle of a BALB/c mouse was peeledinto front and back epidermal sheets, and each of the epidermal sheetswas soaked into Hanks's solution supplemented with 1% trypsin at 37° C.for 30 minutes to 1 hour. The epidermal sheets obtained were placed on amesh, and shaken up and down in Hanks's solution supplemented with 10%FCS. The cells detached from the epidermal sheets were collected, andthen suspended into RPMI solution supplemented with 10% FCS at 10⁶cells/mL. After addition of either a vehicle (DMSO, final concentration:0.1%) or KRN7000 (final concentration: 100 ng/mL), the cells werecultured under 5% CO₂ at 37° C. for 3 days. Thereafter, the cells thatwere non-adherent to the dish were collected and layered overLympholite-M. After centrifugation at 1400 rpm for 10 minutes, thelow-density cells were collected, washed with RPMI twice, which wereprovided for use as APCs.

As responder cells, spleen cells were prepared from BALB/c mice in thesame manner as described in Pharmacological Test 3. The responder cellsthus obtained were added to wells of a 96-well plate (2.5×10⁵cells/well), and the APCs were also added thereto for syngeneic MLRassay (4×10⁴, 4/3×10 and 4/9×10⁴ cells/well, respectively). Afterculturing for 2 days, ³H-TdR was added to each well (0.5 μCi/mL). After6 hours, the cells were harvested and determined on the amount of ³H-TdRtaken up into the cells using a liquid scintillation counter. Theresults (mean value and standard deviation of 3 wells) are shown in FIG.8. In FIG. 8, “V-APC” and “KRN-APC” refer to the APCs pretreated withthe vehicle and KRN7000, respectively.

As shown in FIG. 8, the murine epidermis-derived APCs stimulated withKRN (KRN-APCs) showed a more remarkable syngeneic MLR enhancing effectcompared to those stimulated with the vehicle (V-APCs).

[Pharmacological Test 8] APC Therapy With Murine Spleen-Derived APCspretreated With KRN7000 and Tumor Antigen

The experiment was performed with BDF1 mice (6-week-old, female), whichwere divided into groups consisting of 6 mice. Murine melanoma B16 cellswere implanted subcutaneously into each mouse at a level of 1.5×10⁶cells/mouse (implantation day: day 0). Several types of pretreated APCswere prepared in the same manner as in Pharmacological Test 3, in whichAPCs were pretreated with a vehicle (DMSO, final concentration: 0.1%)(V-APC), with both the vehicle (DMSO, final concentration: 0.1%) and aB16-tumor cell lysate (V-T-APC), with KRN7000 (final concentration: 100ng/mL) (KRN-APC), and with both KRN7000 (final concentration: 100 ng/mL)and a B16-tumor cell lysate (KRN-T-APC), respectively. Each of thepretreated APCs was injected intravenously into the mice at a dose of5×10⁵ cells/mouse on day 1. As a positive control, KRN7000 was injectedintravenously into the mice at a dose of 100 μg/kg on days 1, 5 and 9.The volume of the subcutaneous tumor in each mouse was measured. Theresults (the average of the tumor volumes of 6 mice) are shown in FIG.9.

As shown in FIG. 9, little or no tumor growth-suppressive effect wasobserved when V-APCs and V-T-APCs were injected. On the contrary, whenKRN-APCs were injected once, a tumor growth-inhibitory effect wasobserved, which was at the same level as that when KRN7000 was injected3 times intravenously at a dose of 100 μg/kg. It is interesting to notethat the one injection of KRN-T-APC produced a more remarkable tumorgrowth inhibitory effect than the one injection of KRN-APC or the threeintravenous injections of KRN7000 at a dose of 100 μg/kg.

[Pharmacological Test 9] Effect of KRN7000 on Human PeripheralBlood-Derived APCs

A monocyte fraction was prepared from human peripheral blood mononuclearcells by Percol density-gradient centrifugation. The monocyte fractionwas cultured in the presence or absence of GM-CSF (50 ng/mL) and IL-4(100 ng/mL.), and simultaneously either a vehicle (v; DMSO, 0.1%) orKRN7000 (KRN; 100 ng/mL) was added thereto. After culturing for 3 days,the cells were collected and washed with the medium 3 times to giveV-APC, KRN-APC, V-APC-GM and KRN-APC-GM (where “GM” refers to the APCscultured in the presence of GM-CSF and IL-4), which were then providedfor use as APCs. As responder cells, human peripheral blood-derived FcR⁻cells were used to perform allogeneic MLR assay.

As shown in FIG. 10, all of the APCs enhanced the allogeneic MLRresponse in a manner dependent on the number of the APCs, however,KRN-APC and KRN-APC-GM showed a clearly stronger stimulatory effect onthe proliferation of responder cells compared to V-APC and V-APC-GM,respectively. Furthermore, it was also found that the effect ofKRN-APC-GM was stronger than KRN-APC. It is assumed that the suppressedproliferation of the responder cells by the addition of KRN-APC-GM in anamount of 20000 cells/well is due to the over growth of the respondercells.

[Pharmacological Test 101 Effort of XRN7000 on Human PeripheralBlood-derived APC

To study the effect of long-term culture of APC, the followingexperiment was performed. A monocyte fraction was prepared from humanperipheral mononuclear cells by Percol density-gradient centrifugation.The monocyte fraction was cultured in the presence of GM-CSF (50 ng/mL)and IL-4 (100 ng/mL), and simultaneously either a vehicle (V; 0.1% DMSO)or KRN7000 (KRN; 100 ng/mL) was added thereto. After culturing for 7days, the cells were recovered and washed with the medium 3 times togive V-APC-GM and KRN-APC-GM, respectively, which were provided for useas APCs. As responder cells, human peripheral blood-derived FcR⁻ cellswere used to perform autologous MLR assay.

As shown in FIG. 11, V-APC-GM did not stimulate the proliferation of theresponder cells, whereas KRN-APC-GM enhanced the autologous MLR responsein a manner dependent on the number of the APCs.

(Pharmacological Test 11] Effect of KRN7000 on Human PeripheralBlood-Derived APC

To study the effect of long-term culture of APC, the followingexperiment was performed. A monocyte fraction was prepared from humanperipheral mononuclear cells by Percol density-gradient centrifugation.The monocyte fraction was cultured in the presence of GM-CSF (50 ng/mL)and IL-4 (100 ng/mL) with or without monocyte conditioned medium (MCM)prepared by the method of Bender et al (Bender A. et al., 1996, J.Immunol. Method., 196, 121). Simultaneously either a vehicle (V; 0.1%DMSO) or KRN7000 (KRN; 100 ng/mL) was added to the fraction. Afterculturing for 11 days, the cells were recovered and washed with themedium 3 times to give V-APC-GM (MCM−), KRN-APC-GM (MCM−), V-APC-GM(MC-M+) and KRN-APC-GM (MCM+), respectively, which were provided for useas APCs. As responder cells, human peripheral blood-derived FcR⁻ cellswere used to perform autologous MLR assay.

As shown in FIG. 12, V-APC-GM (MCM−) did not stimulate the proliferationof the responder cells, whereas KRN-APC-GM (MCM−) enhanced theautologous MLR response in a manner dependent on the number of the APCs.Both V-APC-GM (MCM+) and KRN-APC-GM (MCM+) enhanced the autologous MLRresponse in a manner dependent on the number of the APCs. The effect ofKRN-APC-GM (MCM+) was stronger than that of V-APC-GM (MCM+).

From the results of Pharmacological Tests 9, 10 and 11 above, it hasbeen found that KRN7000 can activate the APCs from human peripheralblood by various methods.

[Pharmacological Test 12] Effort of KRN7000 on Human Umbilical CordBlood-Derived APC

A monocyte fraction was prepared from human umbilical cord bloodmononuclear cells by Percol density-gradient centrifugation. Themonocyte fraction was cultured in the presence of GM-CSF (50 ng/mL) andIL-4 (100 ng/mL), and simultaneously either a vehicle (V; 0.1% DMSO) orKRN7000 (KRN; 100 ng/mL) was added thereto. After culturing for 3 days,the cells were recovered and washed with the medium 3 times to giveV-APC-GM and KRN-APC-GM, respectively, which were provided for use asAPCs. As response cells, human umbilical cord blood-derived FcR⁻ cellswere used for autologous MLR assay.

As shown in FIG. 13, V-APC-GM did not stimulate the proliferation of theresponder cells, whereas KRN-APC-GM enhanced the autologous MLR responsein a manner dependent on the number of the APCs. It is assumed that thesuppressed proliferation of the responder cells by the addition ofKRN-APC-GM in an amount of 20,000 cells/well is due to the over growthof the responder cells.

This result shows that KRN7000 can activate the APCs derived from humanumbilical cord blood by various methods.

(Pharmacological Test 13] Effect of α-galactosylceramides on MurineSpleen-Derived APC

To demonstrate that the α-galactosylceramide derivatives of the presentinvention other than KRN7000 also have an APC-activating effect, thefollowing pharmacological test was performed.

The dendritic cell-rich APCs were prepared from murine spleen in thesame manner as described in Pharmacological Test 3. Briefly, the APCswere treated with any one of KRN7000 (compound No.14 of the invention),AGL512 (Compound No.10 of the invention), AGL525 (Compound No.16 of theinvention), AGL506 (Compound No.1 of the invention), AGL514 (CompoundNo.2 of the invention), AGL571 (Compound No.5 of the invention) and avehicle (DMSO, final concentration: 0.1%) at the final concentration of100 ng/mL. Each of the APCs thus treated (1×10⁴ cells/well) and theresponder cells (2.5×10⁵ cells/well) were added to wells of a 96-wellplate and then subjected to syngeneic MLR assay. After culturing for 2days, ³H-TdR was added to each well (0.5 μCi/well). After 8 hours, thecells were harvested, and the amount of the ³H-TdR taken up into thecells was determined using a liquid scintillation counter. The resultsare shown in FIG. 14.

In FIG. 14, “V-APC”, “KRN-APC”, “512-APC”, “525-APC”, “506-APC”,“514-APC” and “571-APV” refer to the APCs pretreated with the vehicle,KRN7000, AGL-512, AGL-525, AGL-506, AGL-514 and AGL-571, respectively.

As shown in FIG. 14, the APCs pretreated with α-galactosylceramidederivatives showed a remarkable syngeneic MLR enhancing effect.

This result shows that the compounds of the present invention(α-galactosylceramides and α-glucosylceramides) other than KRN7000 alsohave an effect of activating the dendritic cell-rich APCs derived frommurine spleen (i.e., an APC function-enhancing effect).

1-23. (canceled)
 24. A method for activating a human antigen-presentingcell, comprising culturing human dendritic cells in vitro with at leastone of the glycoside compounds represented by formula (A) or saltsthereof:

wherein R₁ is H or OH; X is an integer of from 7 to 25; R₂ is asubstituent defined by any one of the following (a) to (e): (a)—CH₂(CH₂)_(Y)CH₃; (b) —CH(OH)(CH₂)_(Y)CH₃; (c) —CH(OH)(CH2)_(v)CH(CH₃)₂;(d) —CH═CH(CH2)_(Y)CH₃; and (e) —CH(OH)(CH₂)_(Y)CH(CH₃)CH₂CH₃ wherein Yis an integer of from 5 to 17; R₃ is H; R₄ is OH; R₅ is OH; R₆ is H; oneof R₇ and R₈ is H and the other is OH; and R₉ is H, CH₃ or CH₂OH. 25.The method of claim 24, wherein the human dendritic cells are obtainedby culturing human monocytes in vitro in the presence of GM-CSF andIL-4,
 26. The method of claim 25, wherein the human monocyte is preparedfrom human peripheral blood.
 27. The method of claim 25, wherein thehuman monocyte is prepared from human umbilical cord blood.
 28. Themethod of claim 25, wherein the human monocyte is prepared from a humanbone marrow cell.
 29. The method of claim 25, wherein the human monocyteis prepared from human epidermis.
 30. The method of claim 24 or 25,wherein the glycoside compound is a compound represented by formula (B):

wherein: R₁ is H or OH; X is an integer of from 7 to 25; R₂ is asubstituent defined by any one of the following (a) to (e): (a)—CH₂(CH₂)_(Y)CH₃; (b) —CH(OH)(CH₂)_(Y)CH₃; (c) —CH(OH)(CH₂)_(v)CH(CH₃)₂;(d) —CH═CH(CH₂)_(Y)CH₃; and (e) —CH(OH)(CH₂)_(Y)CH(CH₃)CH₂CH₃ wherein Yis an integer of from 5 to 17; R₃ to R₉ are substituents defined by anyone of the following (i) to (ii): (i) R₃, R₆ and R₈ are each H; R₄ isOH; R₅ is OH; R₇ is OH; and R₉ is H, CH₃, or CH₂OH; (ii) R₃, R₆ and R₇are each H; R₄, R₅ and R₉ are as defined as in (i); and R₆ is OH. 31.The method of claim 24 or 25, wherein the glycoside compound is acompound represented by formula (B):

wherein: R₁ is H or OH; X is an integer of from 7 to 25; R₂ is asubstituent defined by any one of the following (a) to (e): (a)—CH₂(CH₂)_(Y)CH₃; (b) —CH(OH)(CH₂)_(Y)CH₃; (c) —CH(OH)(CH₂)_(Y)CH(CH₃)₂;(d) —CH═CH(CH₂)_(Y)CH₃; and (e) —CH(OH)(CH₂)_(Y)CH(CH₃)CH₂CH₃ wherein Yis an integer of from 5 to 17; R₃, R₆, and R₈ are each H; R₄, R₅, and R₇are each OH; and R₉ is CH₂OH.
 32. The method of claim 24 or 25, whereinthe glycoside compound is a compound represented by formula (B):

wherein R₁ is H or OH; X is an integer of from 7 to 25; R₂ is asubstituent defined by any one of the following: (b)—CH(OH)(CH₂)_(Y)CH₃; (c) —CH(OH)(CH₂)_(Y)CH(CH₃)₂; and (d)—CH(OH)(CH₂)_(Y)CH(CH₃)CH₂CH₃; wherein Y is an integer of from 5 to 17;and R₃, R₆ and R₈ are each H; R₄, R₅ and R₇ are each OH; and R₉ isCH₂OH.
 33. The method of claim 24 or 25, wherein the glycoside compoundis a compound represented by formula (B):

wherein X is an integer of from 7 to 25; R₁ is H; R₂ is—CH(OH)(CH₂)_(Y)CH₃ where Y is an integer of from 5 to 17; H₃, R₆ and R₈are each H; R₄, R₅ and R₇ are each OH; and R₉ is CH₂OH.
 34. The methodof claim 24 or 25, wherein the glycoside compound is a compoundrepresented by formula (B):

wherein X is an integer of from 7 to 25; R₁ is H; R₂ is—CH(OH)(CH₂)_(Y)CH₃ where Y is an integer of from 5 to 17 and where theOH group is of R configuration; and R₃, R₆ and R₈ are each H; R₄, R₅ andR₇ are each OH; and R₉ is CH₂OH.
 35. The method of claim 24 or 25,wherein the glycoside compound is a compound represented by formula (B):

wherein X is from 21 to 25 and Y is an integer of from 11 to 15; R₁ isH; R₂ is —CH(OH)(CH₂)_(Y)CH₃ where Y is an integer of from 5 to 17 andwhere the OH group is of R configuration; and R₃, R₆ and R₈ are each H;R₄, R₅ and R₇ are each OH; and R₉ is CH2OH.
 36. The method of claim 24or 25, wherein the glycoside compound is selected from the groupconsisting of: (2S, 3S,4R)-1-(α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecanediol;(2S,3R)-1-(α-D-galactopyranosyloxy)-2-[(R)-2-hydroxytetracosanoylamino]-3-octadecanol;(2S, 3R)-1-(α-D-galactopyranosyloxy)-2-tetracosanoylamino-3-octadecanol;(2S,3R)-1-(6′-deoxy-α-D-galactopyranosyloxy)-2-tetracosanoylamino-3-octadecanediol;(2S, 3S,4R)-1-(α-D-galactopyranosyloxy)-2-[(R)-2-hydroxytetracosanoylamino]-3,4-octadecanediol;and (2S, 3S,4R)-1-(α-D-galactopyranosyloxy)-2-tetracosanoylamino-3,4-octadecanediol.